Anti-tumor Effect of Ginkgo biloba Exocarp Extracts on B16 Melanoma Bearing Mice Involving P I3K/Akt/HIF-1α/VEGF Signaling Pathways.

The objective of this study is to investigate the anti-tumor effect of Ginkgo biloba exocarp extracts (GBEE) on B16 melanoma bearing mice and its related molecular mechanisms. The B16-F10 melanoma solid tumor model was established in C57BL/6J mice. The tumor-bearing mice were treated with GBEE (50, 100, 200 mg/kg), taking cis-Dichlorodiamineplatinum (Ⅱ) (DDP, 3 mg/kg) as positive control and normal saline (NS) as model control. After 17 days of administration, the transplanted tumors was stripped and weighed, and the inhibition rate was calculated. Quantitative Reverse Transcription Polymerase chain reaction (qRT-PCR), Western Blot and immunohistochemistry were applied to detect mRNA and protein levels of related factors in B16 transplanted tumor tissues. The results indicated that GBEE (50, 100, 200 mg/kg) inhibited the growth of B16 transplanted solid tumor in C57BL/6J mice. Meanwhile, it inhibited the expression of CD34 and reduced microvessel density (MVD) in a dose-dependent manner. Moreover, GBEE dose-dependently down-regulated the mRNA and protein levels of hypoxia inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF), and vascular endothelial growth factor receptor 2 (VEGFR2). The phosphoinositide 3-kinase (PI3K) and protein kinase B (Akt) proteins were not changed obviously, but the protein levels of p-PI3K and p-Akt were down-regulated. Overall, the inhibitory effect of GBEE on the growth of B16 melanoma transplant tumor in mice is related to inhibiting angiogenesis, and the mechanism involves the regulation of PI3K/Akt/ HIF-lα/VEGF signaling pathway.

Clinical features and dysfunctions of iron metabolism in Parkinson disease patients with hyper echogenicity in substantia nigra: a cross-sectional study.

BACKGROUND: Transcranial ultrasound is a useful tool for providing the evidences for the early diagnosis and differential diagnosis of Parkinson disease (PD). However, the relationship between hyper echogenicity in substantia nigra (SN) and clinical symptoms of PD patients remains unknown, and the role of dysfunction of iron metabolism on the pathogenesis of SN hyper echogenicity is unclear. METHODS: PD patients was detected by transcranial sonography and divided into with no hyper echogenicity (PDSN-) group and with hyper echogenicity (PDSN+) group. Motor symptoms (MS) and non-motor symptoms (NMS) were evaluated, and the levels of iron and related proteins in serum and cerebrospinal fluid (CSF) were detected for PD patients. Data comparison between the two groups and correlation analyses were performed. RESULTS: PDSN+ group was significantly older, and had significantly older age of onset, more advanced Hohen-Yahr stage, higher SCOPA-AUT score and lower MoCA score than PDSN- group (P < 0.05). Compared with PDSN- group, the levels of transferrin and light-ferritin in serum and iron level in CSF were significantly elevated (P < 0.05), but ferroportin level in CSF was significantly decreased in PDSN+ group (P < 0.05). CONCLUSIONS: PD patients with hyper echogenicity in SN are older, at more advanced disease stage, have severer motor symptoms, and non-motor symptoms of cognitive impairment and autonomic dysfunction. Hyper echogenicity of SN in PD patients is related to dysfunction of iron metabolism, involving increased iron transport from peripheral system to central nervous system, reduction of intracellular iron release and excessive iron deposition in brain.

Minimally Toxic Dose of Lipopolysaccharide and α-Synuclein Oligomer Elicit Synergistic Dopaminergic Neurodegeneration: Role and Mechanism of Microglial NOX2 Activation.

The aim of this study is to investigate the role and mechanism of microglial NOX2 activation in minimally toxic dose of LPS and Syn-elicited synergistic dopaminergic neurodegeneration. NOX2+/+ and NOX2-/- mice and multiple primary cultures were treated with LPS and/or Syn in vivo and in vitro. Neuronal function and morphology were evaluated by uptake of related neurotransmitter and immunostaining with specific antibody. Levels of superoxide, intracellular reactive oxygen species, mRNA and protein of relevant molecules, and dopamine were detected. LPS and Syn synergistically induce selective and progressive dopaminergic neurodegeneration. Microglia are functionally and morphologically activated, contributing to synergistic dopaminergic neurotoxicity elicited by LPS and Syn. NOX2-/- mice are more resistant to synergistic neurotoxicity than NOX2+/+mice in vivo and in vitro, and NOX2 inhibitor protects against synergistic neurotoxicity through decreasing microglial superoxide production, illustrating a critical role of microglial NOX2. Microglial NOX2 is activated by LPS and Syn as mRNA and protein levels of NOX2 subunits P47and gp91 are enhanced. Molecules relevant to microglial NOX2 activation include PKC-σ, P38, ERK1/2, JNK, and NF-КBP50 as their mRNA and protein levels are elevated after treatment with LPS and Syn. Combination of exogenous and endogenous environmental factors with minimally toxic dose synergistically propagates dopaminergic neurodegeneration through activating microglial NOX2 and relevant signaling molecules, casting a new light for PD pathogenesis.

Iron deposition in substantia nigra: abnormal iron metabolism, neuroinflammatory mechanism and clinical relevance.

Parkinson disease (PD) is associated with multiple factors, including iron, which is demonstrated to deposit excessively in PD brains. We detected iron deposition by susceptibility weighted image (SWI) and measured the levels of iron metabolism-related proteins and inflammatory factors in cerebrospinal fluid (CSF) and serum of PD patients and control subjects. Clinical symptoms of PD were evaluated by series of rating scales. Relationships among above factors were analyzed. Results showed that corrected phase (CP) value of substantia nigra (SN) was significantly decreased in PD group compared to control group, hence, SN was the main region with excessive iron deposition. In PD group, ferritin was significantly elevated in CSF and reduced in serum compared to control group, and levels of ferritin in CSF and serum were both significantly and positively correlated with CP value of SN, thus, abnormal iron metabolism in central and peripheral systems was associated with iron deposition. CP value of SN in PD group was significantly and negatively correlated with interleukin-1ß level in CSF, so interleukin-1ß might be a neuroinflammatory factor produced by excessive iron in SN. Iron deposition in SN was significantly correlated with motor symptoms and part of non-motor symptoms of PD.

Reversal effect and mechanism of Ginkgo biloba exocarp extracts in multidrug resistance of mice S180 tumor cells.

The aim of the present study was to investigate the reversal effect and its related mechanism of Ginkgo biloba exocarp extracts (GBEEs) in obtained multidrug resistance (MDR) of mice S180 tumor cells in vitro and in vivo. In order to simulate the clinical PFC [cis-dichlorodiamineplatinum, cisplatin (DDP) + fluorouracil (FU), FU+cyclophosphamide and cyclophosphamide] scheme, a gradually increasing dose was administered in a phased induction in order to induce S180 cells in vivo and to make them obtain multidrug resistance. The results in vitro demonstrated that GBEE could significantly increase the IC50 of DDP on S180 MDR cells, increase the accumulation of Adriamycin (ADR) and rhodamine 123 (Rho 123), and reduce the efflux of Rho 123 of S180 MDR cells. The results from the in vivo treatment with a combination of GBEE and DDP to S180 MDR ascites tumor in mice demonstrated that each dose of GBEE could effectively reverse the drug-resistance of S180 MDR cells to DDP in order to extend the survival time of mice with ascite tumors and inhibit tumor growth in solid tumor mice. In addition, GBEE effectively inhibited the expression of MDR-1 mRNA and multidrug resistance-associated protein-1 mRNA in S180 MDR cells of ascites tumor in mice and improved the expression levels of cytokines, including interleukin (IL)-3, IL-18 and interferon-γ in the blood serum of S180 MDR tumor-bearing mice. The present study showed that the mechanism of GBEE reversal of MDR may be associated with the inhibition of the functional activity of P-glycoprotein, the downregulation of drug resistance related gene expression of S180 MDR cells and the improvement of the production of related serum cytokines of S180 MDR tumor mice.

Excessive Iron and α-Synuclein Oligomer in Brain are Relevant to Pure Apathy in Parkinson Disease.

OBJECTIVES: To investigate the demographic features, clinical features, and potential mechanism in patients with Parkinson disease (PD) with pure apathy. METHOD: A total of 145 patients with PD without depression and dementia and 30 age-matched controls were consecutively recruited. Patients with PD were evaluated by Apathy Scale (AS), scales for motor symptoms and quality of life. The levels of iron, oxidative and neuroinflammatory factors, α-synuclein oligomer, and dopamine in cerebrospinal fluid (CSF) from patients with PD and controls were detected by enzyme-linked immunosorbent assay, chemical colorimetric method, and high-performance liquid chromatography. Comparisons between PD with pure apathy and with no pure apathy groups and correlation between AS score and the levels of above factors were analyzed. RESULTS: There were 64 (44.14%) cases in PD-apathy group. The PD-apathy group had older age, (97.81 ± 10.82) years versus (61.86 ± 10.80) years, and severer quality of life (P < .05). The PD-apathy and PD without apathy groups presented no remarkable differences in motor symptoms (P > .05). The levels of iron, hydroxyl radical (·OH), hydrogen peroxide (H2O2), and α-synuclein oligomer in CSF in PD-apathy group were significantly higher than that in PD without the apathy group (P < .05). In patients with PD, the AS score was positively correlated with the levels of iron, ·OH, H2O2 and α-synuclein oligomer in CSF (r = 19.838, .063, 1.046, and 0.498, respectively, P < .05). In PD-apathy group, iron level was positively correlated with ·OH level (r = .011, P < .05), and H2O2 level was positively correlated with α-synuclein oligomer level in CSF (r = .045, P < .05). CONCLUSION: Patients with PD had high prevalence of pure apathy. Patients with PD having pure apathy had older age. Pure apathy reduced quality of life for patients without worsening motor function. Excessive iron and α-synuclein oligomer in brain commonly contributed to pure apathy of PD through potential mechanism of oxidative stress.

Investigation on Abnormal Iron Metabolism and Related Inflammation in Parkinson Disease Patients with Probable RBD.

OBJECTIVE: To investigate potential mechanisms involving abnormal iron metabolism and related inflammation in Parkinson disease (PD) patients with probable rapid eye movement sleep behavior disorder (PRBD). METHODS: Total 210 PD patients and 31 controls were consecutively recruited. PD patients were evaluated by RBD Screening Questionnaire (RBDSQ) and classified into PRBD and probable no RBD (NPRBD) groups. Demographics information were recorded and clinical symptoms were evaluated by series of rating scales. Levels of iron and related proteins and inflammatory factors in cerebrospinal fluid (CSF) and serum were detected. Comparisons among control, NPRBD and PRBD groups and correlation analyses between RBDSQ score and levels of above factors were performed. RESULTS: (1) The frequency of PRBD in PD patients is 31.90%. (2) PRBD group has longer disease duration, more advanced disease stage, severer motor symptoms and more non-motor symptoms than NPRBD group. (3) In CSF, levels of iron, transferrin, NO and IL-1ß in PRBD group are prominently increased. RBDSQ score is positively correlated with the levels of iron, transferrin, NO and IL-1ß in PD group. Iron level is positively correlated with the levels of NO and IL-1ß in PD group. (4) In serum, transferrin level is prominently decreased in PRBD group. PGE2 level in PRBD group is drastically enhanced. RBDSQ score exhibits a positive correlation with PGE2 level in PD group. CONCLUSIONS: PRBD is common in PD patients. PRBD group has severer motor symptoms and more non-motor symptoms. Excessive iron in brain resulted from abnormal iron metabolism in central and peripheral systems is correlated with PRBD through neuroinflammation.

Effect and Potential Mechanism of Electroacupuncture Add-On Treatment in Patients with Parkinson's Disease.

Objectives. To explore effectiveness and mechanisms of electroacupuncture (EA) add-on treatment in Parkinson's disease (PD) patients. Methods. Fifty PD patients were randomly assigned to drug plus EA (D + EA) group and drug alone (D) group. Subjects in D + EA group received stimulation in points of bilateral fengfu, fengchi, hegu, and central dazhui. Participants were evaluated by scales for motor and nonmotor symptoms. Levels of neuroinflammatory factors and neurotransmitters in serum were detected. Results. EA add-on treatment remarkably reduced scores of Unified Parkinson's Disease Rating Scale (UPDRS) III and its subitems of tremor, rigidity, and bradykinesia and conspicuously decreased UPDRS III scores in patients with bradykinesia-rigidity and mixed types and mild severity. Depression and sleep disturbances were eased, which were reflected by decreased scores of Hamilton Depression Rating Scale, Pittsburgh Sleep Quality Index, and elevated noradrenaline level. Effects of EA add-on treatment on motor symptoms and sleep disturbances were superior to drug alone treatment, markedly improving life quality of PD patients. EA add-on treatment decreased nitric oxide level in serum. Conclusions. EA add-on treatment is effective on most motor symptoms and some nonmotor symptoms and is particularly efficacious in PD patients at early stage. Antineuroinflammation may be a mechanism of EA add-on treatment.

Parkinson disease with REM sleep behavior disorder: features, α-synuclein, and inflammation.

OBJECTIVES: To investigate clinical features and potential mechanisms involving α-synuclein oligomer and inflammation in patients with Parkinson disease (PD) and probable REM sleep behavior disorder (PRBD). METHODS: We used the REM Sleep Behavior Disorder Screening Questionnaire (RBDSQ) to evaluate patients with PD and classified each as PRBD or not probable (NPRBD). Data collection included demographic information and evaluation of clinical symptoms using a series of rating scales. We tested for α-synuclein oligomer and inflammatory factors in CSF and serum. Data analyses included comparisons between PRBD and NPRBD groups and correlation analyses among RBDSQ score and levels of the above factors. RESULTS: The frequency of PRBD in patients with PD was 30.67%. The PRBD group had longer disease duration, more advanced disease stage, more severe motor symptoms, and other more severe nonmotor symptoms, including depression, anxiety, and fatigue. Levels of α-synuclein oligomer in CSF and serum in the PRBD group were elevated compared with NPRBD and control groups. RBDSQ score was increased with the elevated α-synuclein oligomer level in CSF, interleukin 1ß and nitric oxide levels in CSF, and prostaglandin E2 level in serum in the PD group. The level of α-synuclein oligomer in CSF was enhanced with the deterioration of motor symptoms, and the elevated levels of interleukin 1ß, nitric oxide, and tumor necrosis factor α in CSF in the PRBD group. CONCLUSIONS: PRBD is common in patients with PD, especially those with longer disease duration and more severe motor and nonmotor symptoms. Elevated α-synuclein levels in CSF and serum may be correlated with PRBD through inflammation in central and peripheral nervous systems.

Role and mechanism of microglial activation in iron-induced selective and progressive dopaminergic neurodegeneration.

Parkinson's disease (PD) patients have excessive iron depositions in substantia nigra (SN). Neuroinflammation characterized by microglial activation is pivotal for dopaminergic neurodegeneration in PD. However, the role and mechanism of microglial activation in iron-induced dopaminergic neurodegeneration in SN remain unclear yet. This study aimed to investigate the role and mechanism of microglial ß-nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) activation in iron-induced selective and progressive dopaminergic neurodegeneration. Multiple primary midbrain cultures from rat, NOX2+/+ and NOX2-/- mice were used. Dopaminergic neurons, total neurons, and microglia were visualized by immunostainings. Cell viability was measured by MTT assay. Superoxide (O2·-) and intracellular reactive oxygen species (iROS) were determined by measuring SOD-inhibitable reduction of tetrazolium salt WST-1 and DCFH-DA assay. mRNA and protein were detected by real-time PCR and Western blot. Iron induces selective and progressive dopaminergic neurotoxicity in rat neuron-microglia-astroglia cultures and microglial activation potentiates the neurotoxicity. Activated microglia produce a magnitude of O2·- and iROS, and display morphological alteration. NOX2 inhibitor diphenylene iodonium protects against iron-elicited dopaminergic neurotoxicity through decreasing microglial O2·- generation, and NOX2-/- mice are resistant to the neurotoxicity by reducing microglial O2·- production, indicating that iron-elicited dopaminergic neurotoxicity is dependent of NOX2, a O2·--generating enzyme. NOX2 activation is indicated by the increased mRNA and protein levels of subunits P47 and gp91. Molecules relevant to NOX2 activation include PKC-σ, P38, ERK1/2, JNK, and NF-КBP65 as their mRNA and protein levels are enhanced by NOX2 activation. Iron causes selective and progressive dopaminergic neurodegeneration, and microglial NOX2 activation potentiates the neurotoxicity. PKC-σ, P38, ERK1/2, JNK, and NF-КBP65 are the potential molecules relevant to microglial NOX2 activation.

Sleep disorders in Parkinson's disease: clinical features, iron metabolism and related mechanism.

OBJECTIVE: To investigate clinical features, iron metabolism and neuroinflammation in Parkinson's disease (PD) patients with sleep disorders (SD). METHODS: 211 PD patients were evaluated by Pittsburgh Sleep Quality Index (PSQI) and a body of scales for motor symptoms and non-motor symptoms. 94 blood and 38 cerebral spinal fluid (CSF) samples were collected and iron and its metabolism-relating proteins, neuroinflammatory factors were detected and analyzed. RESULTS: 136 cases (64.5%) of PD patients were accompanied by SD. Factor with the highest score in PSQI was daytime dysfunction. Depression, restless leg syndrome, autonomic symptoms and fatigue contributed 68.6% of the variance of PSQI score. Transferrin level in serum and tumor necrosis factor-α level in CSF decreased, and the levels of iron, transferrin, lactoferrin and prostaglandin E2 in CSF increased in PD patients with SD compared with those without SD. In CSF, prostaglandin E2 level was positively correlated with the levels of transferrin and lactoferrin, and tumor necrosis factor-α level was negatively correlated with the levels of iron, transferrin and lactoferrin in CSF. CONCLUSIONS: Depression, restless leg syndrome, autonomic disorders and fatigue are the important contributors for the poor sleep in PD patients. Abnormal iron metabolism may cause excessive iron deposition in brain and be related to SD in PD patients through dual potential mechanisms, including neuroinflammation by activating microglia and neurotoxicity by targeting neurons. Hence, inhibition of iron deposition-related neuroinflammation and neurotoxicity may cast a new light for drug development for SD in PD patients.